Parasitic nematodes cause considerable morbidity in humans. Lymphatic filariasis, river blindness, and hookworms infect hundreds of millions and Ascaris alone infects over 1 billion people. The socioeconomic effects caused by these parasites are severe and present a major obstacle in facilitating medical and economic improvements in many parts of the world. Mechanisms of gene expression in parasitic nematodes are poorly understood. Trans-splicing is a major mechanism of gene expression in parasitic nematodes accounting for greater than or equal to 70 percent of the expression and maturation of nematode mRNAs. Spliced leader (SL) trans-splicing is an RNA processing event that forms the 5' termini of mature mRNAs by accurately joining a small, separately transcribed exon (the SL) to the 5' end of pre- mRNAs. The functional significance of trans-splicing in parasitic nematodes remains unknown. We have developed novel strategies to introduce and express nucleic acids in Ascaris embryos to facilitate analysis of gene expression and trans-splicing. With our development of these molecular genetic tools, Ascaris embryos provide an excellent model for analyzing parasitic nematode gene expression. Moreover, it is now possible for the first time to address the functional significance of SL addition in vivo. Using biolistic introduction of luciferase reporter mRNAs into Ascaris embryos, we will examine the kinetics of luciferase activity to evaluate translational efficiency and functional mRNA half-life to test several hypotheses on trans-splicing including: 1) does trans- splicing addition of a leader sequence and unique cap play an important role in mRNA metabolism and 2) does the process of SL addition serve to trim mRNAs, remove inhibitory sequences, produce an optimal translation initiation context or distance from the 5' end of the mRNA to the initiator AUG. In addition, using luciferase reporters and DNA constructs we will test the hypothesis that trans-splicing in parasitic nematodes can serve to functionally resolve polycistronic mRNAs. Other major goals are to examine the role of exon determinants on trans-splicing efficiency and to further develop biolistic methods to directly analyze RNA processing in vivo. The proposed studies will address several outstanding hypotheses regarding trans-splicing and provide information on the functional significance of a major mechanism of gene expression in a model parasitic nematode. These studies may provide insight into the development of novel and cheaper therapeutic agents or vaccine candidates against a broad spectrum of parasites including other trans-splicing organisms such as flatworms and kinetoplastida.